Hemoglobin-oxygen equilibrium curves measured during enzymatic oxygen consumption.
A rapid, new method to measure hemoglobin-oxygen equilibrium curves is described using the protocatechuic acid/protocatechuic acid 3,4-dioxygenase system [C. Bull and D.P. Ballou (1981) J. Biol. Chem. 256, 12673-12680] to deoxygenate hemoglobin solutions enzymatically. The reaction is followed by simultaneous measurements of hemoglobin spectra using a diode array spectrophotometer and oxygen tensions using a polarographic O2 microelectrode. Multicomponent analysis allows the determination of fractions of oxyhemoglobin, deoxyhemoglobin, and high-spin and low-spin methemoglobins in each spectrum collected as the reaction proceeds. Fractional saturation as a function of oxygen partial pressure is calculated as the ratio of oxyhemoglobin to oxy- plus deoxyhemoglobin. Several advantages are offered by this method: (i) Hemoglobin-O2 binding curves are obtained rapidly and reproducibly; (ii) the speed of the reaction limits methemoglobin formation by autooxidation; (iii) there is no gas-liquid interface, eliminating protein denaturation at the surface; and (iv) direct calculations of fractional saturation are made using spectral analysis, thus avoiding the assumption of a linear transition between deoxy- and oxyhemoglobin.
Vandegriff
K D
KD
Department of Medicine, School of Medicine, University of California, San Diego, USA.
Rohlfs
R J
RJ
Magde
M D
MD
Winslow
R M
RM
D005069:Evaluation Studies as Topic; D006454:Hemoglobins / Q000032:analysis*; D006801:Humans; D062385:Hydroxybenzoates; D008706:Methemoglobin / Q000032:analysis; D008839:Microelectrodes; D010100:Oxygen / Q000032:analysis*; D010101:Oxygen Consumption; D010108:Oxyhemoglobins / Q000032:analysis; D011048:Polarography; D011520:Protocatechuate-3,4-Dioxygenase; D012996:Solutions; D013053:Spectrophotometry
D006454:Hemoglobins; D062385:Hydroxybenzoates; D010108:Oxyhemoglobins; D012996:Solutions; C016484:hemoglobin, stroma free; C009091:protocatechuic acid; C100003:deoxyhemoglobin; D008706:Methemoglobin; D011520:Protocatechuate-3,4-Dioxygenase; D010100:Oxygen